This invention relates to the purification and characterization of factors that substantially alter tumor necrosis factor (TNF) receptor (TNF-R) releasing enzyme (TRRE) activity, and methods of use thereof. Modulation of TRRE levels indirectly modulates effective levels of TNF. The invention further relates to methods of treatment of pathological conditions caused or exacerbated by altered levels or activity of TNF such as inflammatory conditions including autoimmune diseases, infections, septic shock, obesity, cachexia, and conditions that are associated with decreased effective levels or activity of TNF such as cancer.
Tumor necrosis factor (TNF or TNF-xcex1) and lymphotoxin (LT or TNF-xcex2) are related cytokines that share 40 percent amino acid (AA) sequence homology. Old (1987) Nature 330:602-603. These cytokines are released mainly by macrophages, monocytes and natural killer (NK) cells in response to broad immune reactions. Gorton and Galli (1990) Nature 346:274-276; and Dubravec et al. (1990) Proc. Natl. Acad. Sci. USA 87:6758-6761. Although initially discovered as agents inducing hemorrhagic necrosis of tumors, these cytokines have been shown to have essential roles in both the inductive and effector phases of immune reactions and inflammation. The two cytokines cause a broad spectrum of effects on cells in vitro and tissues in vivo, including: (i) vascular thrombosis and tumor necrosis; (ii) inflammation; (iii) activation of macrophages and neutrophils; (iv) leukocytosis; (v) apoptosis; and (vi) shock. Beretz et al. (1990) Biorheology 27:455-460; Driscoll (1994) Exp. Lung Res. 20:473-490; Ferrante (1992) Immunol. Ser. 57:417-436; Golstein et al. (1991) Immunol. Rev. 121:29-65; and van der Poll and Lowry (1995) Shock 3:1-12. For a review of the mechanism of action of TNF, see Massague (1996) Cell 85:947-950. TNF has been associated with a variety of disease states including various forms of cancer, arthritis, psoriasis, endotoxic shock, sepsis, autoimmune diseases, infections, obesity, and cachexia. Attempts have been made to alter the course of a disease by treating the patient with TNF inhibitors. These attempts have met with varying degrees of success. For example, oxpentifylline did not alter the course of Crohn""s disease, a chronic inflammatory bowel disease. Bauditz et al. (1997) Gut 40:470-4. However, the TNF inhibitor dexanabinol provided protection against TNF following traumatic brain injury. Shohami et al. (1997) J. Neuroimmun. 72:169-77.
Cachexia is pathological weight loss generally associated with anorexia, weakness, anemia, asthenia, and loss of body lipid stores and skeletal muscle protein. This state often accompanies burns, trauma, infection, and neoplastic diseases. Lawson et al. (1982) Annu. Rev. Nutr. 2:277-301; Argiles et al. (1988) Mol. Cell. Biochem. 81:3-17; and Ogiwara et al. (1994) J. Surg. Oncol. 57:129-133. TNF concentrations are elevated in many patients with cachexia. Scuderi et al. (1986) Lancet 2:1364-65; Grau et al. (1987) Science 237:1210-1212; and Waage et al. (1986) Scand. J Immunol. 24:739-743. TNF inhibits collagen xcex1I gene expression and wound healing in a murine model of cachexia. Buck et al. (1996) Am. J. Pathol. 149:195-204. In septicemia (the invasion of bacteria into the bloodstream), increased endotoxin concentrations may raise TNF levels, causing cachexia. Beutler et al. (1985) Science 229:869-871; Tracey et al. (1987) Nature 330:662-664; and Michie et al. (1988) New Engl. J. Med 318:1481-1486. During cachexia, the loss of white adipose tissue is caused by the decreased activity of lipoprotein lipase (LPL); TNF lowers the activity of this enzyme. Price et al. (1986) Arch. Biochem. Biophys. 251:738-746; Cornelius et al. (1988) Biochem. J. 249:765-769; Fried et al. (1989) J. Lipid. Res. 30:1917-1923; Semb et al. (1987) J. Biol. Chem. 262:8390-8394; and Evans et al. (1988) Biochem. J. 256:1055-1058. Fat tissue loss is also associated with an increase in lipase activity and inhibition of glucose transport; TNF is also linked to both of these changes. Kawakami et al. (1987) J. Biochem. 331-338; Feingold et al. (1992) Endocrinology 130:10-16; and Hauner et al. (1995) Diabetologia 38:764-771. TNF mediates hypertriglyceridaemia associated with cachexia. Dessi et al. (1995) Br. J Cancer 72:1138-43. TNF also participates in the protein wasting, loss of skeletal muscle and loss of nitrogen associated with cachexia. Costelli et al. (1993) J. Clin. Invest. 92:2783-2789; Flores et al. (1989) J. Clin. Invest. 83:1614-1622; Goodman (1991) Am. J. Physiol. 260:E727-730; Zamir et al. (1992) Arch. Surg. 127:170-174; Llovera et al. (1993) J. Natl. Cancer Inst. USA 85:1334-1339; and Garcia-Martinez et al. (1993) FEBS Lett. 323:211-214.
Cachexia is also associated with TNF expression in cancer patients. TNF is linked to the three factors contributing to body weight control: intake, expenditure, and storage of energy. Administration of either TNF or IL-1, for example, induces a decrease in food intake. Rothwell (1993) Int. J. Obesity 17:S98-S101; Arbos et al. (1992) Mol. Cell. Biochem. 1 12:53-59; Fargeas et al. (1993) Gastroenterology 104:377-383; Plata-Salaman et al. (1994) Am. J. Physiol. 266:R1711-1715; Schwartz et al. (1995) Am. J. Physiol. 269:R949-957; and Oliff et al. (1987) Cell 50:555-563. Interestingly, TNF may have key roles in both extremes of weight problems. Abnormalities in its activity may lead to obesity; changes in its production result in the opposite effect, cachexia. Argilxc3xa9s et al. (1997) FASEB J. 11:743-751.
TNF has additional, related roles. It is involved in thermogenesis, particularly nonshivering thermogenesis in brown adipose tissue (BAT), a tissue with an elevated level in cachexia. Nicholls (1983) Biosci. Rep. 3:431-441; Rothwell (1993) Int. J. Obesity 17:S98-S101; Bianchi et al. (1989) Horm. Metab. Res. 21:1 1; and Oudart et al. (1995) Can. J. Physiol. Pharmacol. 73:1625-1631. TNF has also been implicated in non-insulin-dependent (type II) diabetes. Hotamisligil et al. (1995) J. Clin. Invest. 95:2409-2415; Arner (1996) Diabetes Metab. 13:S85-S86; Spiegelman et al. (1993) Cell 73:625-627; Saghizadeh et al. (1996) J. Clin. Invest. 97:1111-16; and Hofmann et al. (1994) Endocrinology 134:264-270.
These data help explain how TNF mediates the opposite effects of obesity and cachexia. TNF has functional similarities to leptin, which has been proposed to be an xe2x80x9cadipostat.xe2x80x9d Zhang et al. (1994) Nature 372:425-432; Phillips et al. (1996) Nature Genet. 13:18-19; and Madej et al. (1995) FEBS Lett. 373:13-18. Like leptin, TNF is expressed and secreted by adipocytes and can travel to the brain. TNF administration also results in an increase in circulating leptin concentrations. Grunfeld et al. (1996) J. Clin. Invest. 97:2152-57. It is possible to reconcile the participation of TNF in obesity and cachexia. TNF can be considered one of many signals coming from adipose tissue that participate in the feedback mechanism that informs the hypothalamic center about the state of the adipocyte energy depot. TNF probably counteracts excessive energy intake and is able to stimulate thermogenesis either directly or by increasing sympathetic activity. TNF released by adipose tissue will also stimulate lipolysis, decrease LPL activity, decrease the expression of the glucose transporter GLUT4, and inhibit lipogenesis in the adipocyte, thus contributing to the maintenance (but not increased fat deposition) of the adipose tissue mass. In cachexia, however, the situation is different. A high production of TNF by activated macrophages (as a result of a tumor or an infection) contributes to anorexia, increased thermogenesis, and adipose tissue dissolution. However, a pathological state can be created where there is an excess of TNF informing the brain that adipose tissue needs dissolution. The two situations can thus be reconciled: in cachexia there is a pathological overproduction of TNF; in obesity, the physiological action of TNF as a signal to control food intake and energy expenditure is impaired. Argilxc3xa9s et al. (1997). FASEB J. 11:743-751.
Attempts have been made to ameliorate the untoward effects of TNF by treatment with monoclonal antibodies to TNF or with other proteins that bind TNF, such as modified TNF receptors. Patients with sepsis or septic shock have been treated with anti-TNF antibodies. Neither coagulation nor the fibrinolytic system was affected by an anti-TNF antibody in a study of patients with sepsis or septic shock. Satal et al. (1996) Shock 6:233-7. Some improvement in the clinical and histopathologic signs of Crohn""s disease were afforded by treatment with anti-TNF antibodies. Neurath et al. (1997) Eur. J. Immun. 27:1743-50; van Deventer et al. (1997) Pharm. World Sci. 19:55-9; van Hogezand et al. (1997) Scand. J. Gastro. 223:105-7; and Stack et al. (1997) Lancet 349:521-4. In the treatment of experimental autoimmune encephalitis (EAE), an animal model of the human disease multiple sclerosis (MS), treatment with TNF-R fusion protein prevents the disease and the accompanying demyelination, suggesting the possible use of this treatment in MS patients. Klinkert et al. (1997) J. Neuroimmun. 72:163-8.
Regulation of TNF expression is being tested in treatment of endotoxic shock. Mohler et al. (1994) Nature 370:218-220. Modulation of TNF-R activity is also being approached by the use of peptides that bind intracellularly to the receptor or other component in the process to prevent receptor shedding. PCT patent publications: WO 95/31544, WO 95/33051; and WO 96/01642. Modulation of TNF-R activity is also postulated to be possible by binding of peptides to the TNF-R and interfering with signal transduction induced by TNF. European Patent Application EP 568 925.
Human TNF and LT mediate their biological activities, both on cells and tissues, by binding specifically to two distinct, although related, glycoprotein plasma membrane receptors of 55 kDa and 75 kDa (p55 and p75 TNF-R, respectively). Holtmann and Wallach (1987) J. Immunol. 139:151-153. The two receptors share 28 percent amino acid (AA) sequence homology in their extracellular domains, which are composed of four repeating cysteine-rich regions. Tartaglia and Goeddel (1992) Immunol. Today 13:151-153. However, the receptors lack significant AA sequence homology in their intracellular domains. Dembic et al. (1990) Cytokine 2:231-237. Due to this dissimilarity, they may transduce different signals and, in turn, exercise diverse functions.
Recent studies have shown that most of the known cellular TNF responses, including cytotoxicity and induction of several genes, may be attributed to p55 TNF-R activation. Engelmann et al. (1990) J. Biol. Chem. 265:1531-1536; Shalaby et al. (1990) J. Exp. Med. 172:1517-1520; and Tartaglia et al. (1991) Proc. Natl. Acad. Sci. USA 88:9292-9296. In addition, the p55 receptor controls early acute graft-versus-host disease. Speiser et al. (1997) J. Immun. 158:5185-90. In contrast, information regarding the biological activities of p75 TNF-R is limited. This receptor shares some activities with p55 TNF-R and specifically participates in regulating proliferation of and secretion of cytokines by T cells. Shalaby et al. (1990); and Gehr et al. (1992) J. Immunol. 149:911-917. Both belong to an ever-increasing family of membrane receptors including low-affinity nerve growth factor receptor (LNGF-R), FAS antigen, CD27, CD30 (Ki-1), CD40 (gp50) and OX 40. Cosman (1994) Stem Cells (Dayt.) 12:440-455; Meakin and Shooter (1992) Trends Neurosci. 15:323-331; Grell et al. (1994) Euro. J. Immunol. 24:2563-2566; Moller et al. (1994) Int. J. Cancer 57:371-377; Hintzen et al. (1994) J. Immunol. 152:1762-1773; Smith et al. (1993) Cell 73:1349-1360; Corcoran et al. (1994) Eur. J. Biochem. 223:831-840; and Baum et al. (1994) EMBO J. 13:3992-4001.
All of these receptors share a repetitive pattern of cysteine-rich domains in their extracellular regions. In accord with the pleiotropic activities of TNF and LT, most human cells express low levels (2,000 to 10,000 receptors/cell) of both TNF-Rs simultaneously. Brockhaus et al. (1990) Proc. Natl. Acad. Sci. USA 87:3127-3131. Expression of TNF-R on both lymphoid and non-lymphoid cells may be up and down-regulated by many different agents, such as bacterial lipopolysaccharide (LPS), phorbol myristate acetate (PMA; a protein kinase C activator), interleukin-1 (IL-1), interferon-gamma (IFN-xcex3) and IL-2. Gatanaga et al. (1991) Cell Immunol. 138:1-10; Yui et al. (1994) Placenta 15:819-835; and Dett et al. (1991) J. Immunol. 146:1522-1526. Although expressed in different proportions, each receptor binds TNF and LT with equally high affinity. Brockhaus et al. (1990); and Loetscher et al. (1990) J. Biol. Chem. 265:20131-20138. Initial studies showed that the complexes of human TNF and TNF-R are formed on the cell membrane, internalized wholly, and then either degraded or recycled. Armitage (1994) Curr. Opin. Immunol. 6:407-413; and Fiers (1991) FEBS Lett. 285:199-212.
TNF binding proteins (TNF-BP) were originally identified in the serum and urine of febrile patients, individuals with renal failure, cancer patients, and even certain healthy individuals. Seckinger et al. (1988) J. Exp. Med. 167:1511-1516; Engelmann et al. (1989) J. Biol. Chem. 264:11974-11980; Seckinger et al. (1989) J. Biol. Chem. 264:11966-11973; Peetre et al. (1988) Eur. J. Haematol. 41:414-419; Olsson et al. (1989) Eur. J. Haematol. 42:270-275; Gatanaga et al. (1990a) Lymphokine Res. 9:225-229; and Gatanaga et al. (1990b) Proc. Natl. Acad. Sci USA 87:8781-8784. In fact, human brain and ovarian tumors produced high serum levels of TNF-BP. Gatanaga et al. (1990a); and Gatanaga et al. (1990b). These molecules were subsequently purified, characterized, and cloned by different laboratories. Gatanaga et al. (1990b); Olsson et al. (1989); Schall et al. (1990) Cell 61:361-370; Nophar et al. (1990) EMBO J. 9:3269-3278; Himmler et al. (1990) DNA Cell Biol. 9:705-715; Loetscher et al. (1990) Cell 61:351-359; and Smith et al. (1990) Science 248:1019-1023. These proteins have been suggested for use in treating endotoxic shock. Mohler et al. (1993) J. Immunol. 151:1548-1561; Porat et al. (1995) Crit. Care Med. 23:1080-1089; Fisher et al. (1996) N. Engl. J. Med. 334:1697-1702; Fenner (1995) Z. Rheumatol. 54:158-164; and Jin et al. (1994) J. Infect. Dis. 170:1323-1326.
Human TNF-BP consist of 30 kDa and 40 kDa proteins found to be identical to the N-terminal extracellular domains of p55 and p75 TNF-R, respectively. The 30 kDa and 40 kDa TNF-BP are thus also termed p55 and p75 sTNF-R, respectively. Studies of these proteins have been facilitated by the availability of human recombinant 30 kDa and 40 kDa TNF-BP and antibodies which specifically recognize each form and allow quantitation by immunoassay. Heller et al. (1990) Proc. Natl. Acad. Sci. USA 87:6151-6155; U.S. Pat. No. 5,395,760; EP 418,014; and Grosen et al. (1993) Gynecol. Oncol. 50:68-77. X-ray structural studies have demonstrated that a TNF trimer binds with three soluble TNF-R (sTNF-R) molecules and the complex can no longer interact with TNF-R. Banner et al. (1993) Cell 73:431-445. The binding of the trimer and sTNF-R, however, is reversible and these reactants are not altered as a result of complex formation. At high molar ratios of sTNF-R to TNF, both recombinant and native human sTNF-R are potent inhibitors of TNF/LT biological activity in vitro as well as in vivo. Gatanaga et al. (1990b); Ashkenazi et al. (1991) Proc. Natl. Acad. Sci. USA 88:10535-10539; Lesslaur et al. (1991) Eur. J. Immunol. 21:2883-2886; Olsson et al. (1992) Eur. J. Haematol. 48:1-9; and Kohno et al. (1990) Proc. Natl. Acad Sci. USA 87:8331-8335.
Increased levels of TNF-R are also associated with clinical sepsis (septic peritonitis), HIV-1 infection, and other inflammatory conditions. Kalinkovich et al. (1995) J. Interferon and Cyto. Res. 15:749-757; Calvano et al. (1996) Arch. Surg. 131:434-437; and Ertel et al. (1994) Arch. Surg. 129:1330-1337. Sepsis, and septic shock affect thousands of patients every year and there is essentially no cure. This lethal syndrome is caused primarily by lipopolysaccharides (LPS) of Gram-negative bacteria and superantigens of Gram-positive bacteria. Clinical symptoms are initiated primarily by the release of endogenous mediators, such as TNF, from activated lymphoid cells into the bloodstream. TNF induces production of a cascade of other cytokines, including IL-1, IFN-xcex3, IL-8, and IL-6. These cytokines, along with other factors, promote the clinical symptoms of shock. Recombinant human sTNF-R is currently being tested in clinical trials to block TNF/LT activity in patients with septic shock and other conditions in which TNF and LT are thought to be pathogenic. Van Zee et al. (1992) Proc. Natl. Acad. Sci. USA 89:4845-4849. Balb/c mice, the primary animal model, and multiple techniques have been used to test the effects of TNF modulators and other treatments on septic peritonitis. Jin et al. (1994) J. Infect. Dis. 170:1323-1326; Mohler et al. (1993) J. Immunol. 151:1548-1561; Porat et al. (1995) Crit. Care Med 23:1080-1089; and Echtenacher et al. (1996) Nature 381:75-77. LPS-induced shock has been shown to be ameliorated by FR167653, a dual inhibitor of IL-1 and TNF production. Yamamoto et al. (1997) Eur. J. Pharmacol. 327:169-174.
While low levels of sTNF-R have been identified in the sera of normal individuals, high levels have been found in the sera of patients with chronic inflammation, infection, renal failure and various forms of cancer. Aderka et al. (1992) Lymphokine Cytokine Res. 11:157-159; Olsson et al. (1993) Eur. Cytokine Netw. 4:169-180; Diez-Ruiz et al. (1995) Eur. J. Haematol. 54:1-8; van Deuren (1994) Eur. J. Clin. Microbiol. Infect. Dis. 13 Suppl. 1:S12-6; Lambert et al. (1994) Nephrol. Dial. Transplant. 9:1791-1796; Halwachs et al. (1994) Clin. Investig. 72:473-476; Gatanaga et al. (1990a); and Gatanaga et al. (1990b). Serum levels of sTNF-R rise within minutes and remain high for 7 to 8 hours after the intravenous injection of human recombinant TNF or IL-2 into human cancer patients. Aderka et al. (1991) Cancer Res. 51:5602-5607; and Miles et al. (1992) Br. J. Cancer 66:1195-1199. It has also been observed that serum sTNF-R levels are chronically elevated in cancer patients and may remain at high levels for years. Grosen et al. (1993). It is clear that sTNF-R are natural inhibitors of these cytokines and regulate their biological activity post secretion. Fusion proteins consisting of a sTNF-R linked to a portion of the human IgG1 have also been developed for treating rheumatoid arthritis and septic shock. Moreland et al. (1997) N. Eng. J. Med. 337:141-7; Abraham et al. (1997) JAMA 277:1531-8.
New evidence has yielded information on cellular regulation of secreted cytokines. The evidence indicates that cells release molecules which resemble or contain the binding site of the specific membrane receptors. Massague and Pandiella (1993) Annu. Rev. Biochem. 62:515-541; and Rose-John and Heinrich (1994) Biochem. J. 300:281-290. These soluble forms specifically bind and, in the appropriate molar ratios, inactivate the cytokine by steric inhibition. Therefore, this may be a general phenomenon responsible for the regulation of cytokines and membrane antigens.
In addition to TNF-R, various types of membrane molecules have both soluble and membrane forms, including (i) cytokine receptors, e.g., IL-1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-7R, IL-9R, granulocyte-colony stimulating factor-R (G-CSF-R), granulocyte-macrophage-colony stimulating factor-R (GM-CSF-R), transforming growth factor-xcex2-R (TGFxcex2-R), platelet-derived growth factor-R (PDGF-R), and epidermal growth factor-R (EGF-R); (ii) growth factors, e.g., TNF-(pro-TNF-xcex1), TGF-xcex1, and CSF-1; (iii) adhesion molecules, e.g., intracellular adhesion molecule-1 (ICAM-1/CD54) and vascular cell membrane adhesion molecule (VCAM-1/CD106); (iv) TNF-R/NGF-R superfamily, e.g., LNGF-R, CD27, CD30, and CD40; and (v) other membrane proteins, e.g. transferrin receptor, CD14 (receptor for LPS and LPS binding protein), CD16 (Fcxcex3RIII), and CD23 (low-affinity receptor for IgE). Colotta et al. (1993) Science 261:472-475; Baran et al. (1988) J. Immunol. 141:539-546; Mosley et al. (1989) Cell 59:335-348; Takaki et al. (1990) EMBO J. 9:4367-4374; Novick et al. (1989) J. Exp. Med. 170:1409-1414; Goodwin et al. (1990) Cell 60:941-95 1; Renauld et al. (1992) Proc. Natl. Acad. Sci. USA 89:5690-5694; Fukunaga et al. (1990) Proc. Natl. Acad. Sci. USA 87:8702-8706; Raines et al. (1991) Proc. Natl. Acad Sci. USA 88:8203-8207; Lopez-Casillas et al. (1991) Cell 67:785-795; Tiesman and Hart (1993) J. Biol. Chem. 268:9621-9628; Khire et al. (1990) Febs. Lett. 272:69-72; Kriegler et al. (1988) Cell 53:45-53; Pandiella and Massague (1991) Proc. Natl. Acad Sci. USA 88:1726-1730; Stein et al. (1991) Oncogene 6:601-605; Seth et al. (1991) Lancet 338:83-84; Hahne et al. (1994) Eur. J. Immunol. 24:421-428; Zupan et al. (1989) J. Biol. Chem. 264:11714-11720; Loenen et al. (1992) Eur. J. Immunol. 22:447-455; Latza et al. (1995) Am. J. Pathol. 146:463-471; Chitambar (1991) Blood 78:2444-2450; Landmann et al. (1992) J. Leukoc. Biol. 52:323-330; Huizinga et al. (1988) Nature 333:667-669; and Alderson et al. (1992) J. Immunol. 149:1252-1257.
In vitro studies with various types of cells have revealed that there are two mechanisms involved in the production of soluble receptors and cell surface antigens. One involves translation from alternatively spliced mRNAs lacking transmembrane and cytoplasmic regions, which is responsible for the production of soluble IL-4R, IL-5R, IL-7R, IL-9R, G-CSF-R, and GM-CSF-R. Rose-John and Heinrich (1994); and Colotta et al. (1993). The other mechanism involves proteolytic cleavage of the intact membrane receptors and antigens, known as shedding. Proteolysis appears to be involved in the production of soluble LNGF-R, TNF-R, CD27, CD30, IL-1R, IL-6R, TGFxcex2-R, PDGF-R, and CD14 (Id.).
Both soluble p55 and p75 TNF-R do not appear to be generated from processed mRNA, since only full length receptor mRNA has been detected in human cells in vitro. Gatanaga et al. (1991). Carboxyl-terminal sequencing of the human soluble p55 TNF-R indicates that a cleavage site may exist between Asn 172 and Val 173. Gullberg et al. (1992) Eur. J. Cell. Biol. 58:307-312. This evidence is supported by the finding that human TNF-R with the mutation at Asn 172 and Val 173 was not released as effectively as native TNF-R on COS-1 cells transduced with cDNA of human TNF-R. Gullberg et al. (1992). The cytoplasmic portion of TNF-R does not appear to play an important role in releasing the soluble receptor forms from transduced COS-1 cells. COS-1 cells release sTNF-R even when transduced with cDNA of human p55 TNF-R which expresses only the extracellular domain but not the cytoplasmic domain. (Id.) sTNF-R shedding is not affected by dexamethasone, gold sodium thiomalate, or prostaglandin E2. Seitz et al. (1997) J. Rheumatology 24:1471-6. Collectively, these data support the concept that human sTNF-R are produced by proteolytic cleavage of membrane TNF-R protein.
PMA is an extremely strong and rapid inducer of TRRE and, indirectly, TNF-R. Basically, PMA is a powerful stimulator of protein kinase C which is anchored inside the cell membrane once activated. Data suggest that (i) TRRE is stored in the cytoplasm very close to the cell membrane ready to be secreted through the protein kinase C cascade by PMA stimulation; (ii) TRRE is a peripheral (or extrinsic) membrane protein which is dissociated from the membrane through the change of interactions with other proteins or with any phospholipid by stimulated protein kinase C; or (iii) TRRE is an integral (or intrinsic) membrane protein which is cleaved and secreted to be an active form after its cytoplasmic portion interacts directly or indirectly with protein kinase C.
TRRE induction by PMA does not require de novo protein synthesis, RNA synthesis and transmission inside the cytoplasm, but only membrane internalization and movement. This is compatible with the data that TRRE was released very quickly by PMA stimulation and halted once PMA was removed. With PMA stimulation, however, TRRE synthesis begins at the same time as TRRE release. After the initial release, TRRE accumulates inside the cell or on the cell surface within 2 hours ready to be secreted by the next stimulation. Evidence for direct cleavage of TNF-R is that the shedding of sTNF-R occurs very quickly (5 minutes), with maximal shedding within 30 minutes.
In addition to PMA, shedding of sTNF-R has been known to be enhanced by several cytokines including TNF, IL-1, IL-6, IL-10 and IFN, leukocyte migration enhancement factors including formyl-methionyl-leucyl-phenylalanine (fMLP) and C5a, and calcium ionophore. Gatanaga (1993) Lymphokine Res. 12:249-253; Porteu (1994) J. Biol. Chem. 269:2834-2840; van der Poll (1995) J. Immunol. 155:5397-5401; Porteu et al. (1991); and Porteu and Natah (1990) J. Exp. Med. 172:599-607. IL-10 and epinephrine induce TRRE in the human monocyte cell line THP-1.
IL-10 is a potent inhibitor of monocyte- and macrophage-functions. Moore (1993) Annu. Rev. Immunol. 11:165-190. IL-10 has anti-inflammatory activity on monocytes and inhibits the release of pro-inflammatory cytokines including TNF and IL-1. Bogdan et al. (1991) J. Exp. Med. 174:1549-1555; Fiorentino et al. (1991) J. Immunol. 147:3815-3822; de Waal Malefyt et al. (1991) J. Exp. Med. 174:1209-1220; Katsikis et al. (1994) J. Exp. Med. 179:1517-1527; Joyce et al. (1994) Eur. J. Immunol. 24:2699-2705; and Simon et al. (1994) Proc. Natl. Acad Sci. USA 91:8562-8566. Elevated levels of IL-10 have been detected in plasma of patients with sepsis, and after administration of LPS to animals. Marchant et al. (1994) Lancet 343:707-708; Derkx et al. (1995) J. Infect. Dis. 171:229-232; Durez et al. (1993) J. Exp. Med 177:551-555; and Marchant et al. (1994) Eur. J. Immunol. 24:1167-1171. In vivo, IL-10 has also been shown to protect mice against endotoxin shock. Gerard et al. (1993) J. Exp. Med. 177:547-550; and Howard et al. (1993) J. Exp. Med. 177:1205-1208. IL-10 leads to increased levels of mRNA for p75 TNF-R, increased release of soluble p75 TNF-R and a concomitant reduction of surface expression of p75 TNF-R on monocytes. Joyce et al. (1994). Thus, IL-10 may be considered to reduce the pro-inflammatory potential of TNF by (i) inhibiting the release of TNF itself, and (ii) down-regulating surface TNF-R expression while (iii) increasing production of sTNF-R capable of neutralizing TNF cytotoxicity. Joyce et al. (1994); and Leeuwenberg et al. (1994) J. Immunol. 152:4036-4043. The data presented herein that IL-10 may induce TRRE activity are consistent with these findings and indicate a newly revealed function of IL-10 as an anti-inflammatory cytokine.
In stressful situations, including endotoxic shock, serum levels of catecholamines and glucocorticoids are elevated chiefly from adrenal medulla and adrenal cortex, respectively, in response to high serum level of adrenocorticotropic hormone (ACTH) throughout the whole body system. TNF also has been implicated in the early metabolic events following stressful situations, and infusion of recombinant TNF in dogs was associated with increase of serum levels of catecholamines, glucocorticoids and glucagon. Tracey et al. (1987) Surg. Gynecol. Obstet. 164:415-422. As a local phenomenon, epinephrine and norepinephrine are found in macrophages which express xcex2-adrenergic receptors and these endogenous catecholamines seem to regulate LPS-induced TNF production in an autocrine fashion in vitro. Hjemdahl et al. (1990) Br. J. Clin. Pharmacol. 30:673-682; Hjemdahl et al. (1990) Br. J. Clin. Pharmacol. 30:673-682; Talmadge et al. (1993) Int. J. Immunopharmacol. 15:219-228; and Spengler et al. (1994) J. Immunol. 152:3024-3031. Exogenous epinephrine and isoproterenol, a specific adrenergic agonist, inhibit the production of TNF from human blood and THP-1 cells stimulated by LPS. Hu et al. (1991) J. Neuroimmunol. 31:35-42; and Severn (1992) J. Immunol. 148:3441-3445.
While epinephrine may be an important endogenous inhibitor of TNF production, especially in sepsis, epinephrine also decreases the number of TNF-R on macrophages. Bermudez et al. (1990) Lymphokine Res. 9:137-145. It has been shown that in trauma patients both p55 and p75 TNF-R levels were significantly elevated along with high serum level of epinephrine within 1 hour of injury. Tan et al. (1993) J. Trauma 34:634-638. These findings are in agreement with the data that epinephrine induced TRRE activity and may lead to the increase of sTNF-R.
In addition to epinephrine, insulin and glucagon have the function to down-regulate TNF-R. Bermudez et al. (1990). Many inflammatory cytokines besides IL-10 may influence the shedding of sTNF-R including TNF, IL-1, IL-6, and IFN for up-regulation and IL-4 for down-regulation. van der Poll et al. (1995); Gatanaga et al. (1993); and Joyce et al. (1994).
Two reports describe the involvement of a metalloprotease in the production of sTNF-R by utilizing a specific metalloprotease inhibitor, TNF-xcex1 protease inhibitor (TAPI). TAPI blocks the shedding of soluble p75 and p55 TNF-R, respectively. Crowe et al. (1995); and Mullberg et al. (1995). Moreover, the processing of pro-TNF on the cell membrane was reported to be dependent on a matrix metalloprotease (MMP)-like enzyme. Gearing et al. (1994); and Gearing et al. (1995). MMPs are a family of structurally related matrix-degrading enzymes that play a major role in tissue remodeling and repair associated with development and inflammation. Matrisian (1990) Trends Genet. 6:121-125; Woessner (1991) FASEB J. 5:2145-2154; and Birkedal-Hansen et al. (1993) Crit. Rev. Oral Biol. Med. 4:197-250. Pathological expression of MMPs is associated with tumor invasiveness, osteoarthritis, atherosclerosis, and pulmonary emphysema. Mignatti et al. (1986) Cell 47:487-498; Khokha (1989) Science 243:947-950; Dean et al. (1989) J. Clin. Invest. 84:678-685; Henney et al. (1991) Proc. Natl. Acad. Sci. USA 88:8154-8158; and Senior et al. (1989) Am. Rev. Respir. Dis. 139:1251-1256. MMPs are Zn2+-dependent enzymes which have Zn2+ in their catalytic domains. Ca2+ stabilizes their tertiary structure significantly. Lowry et al. (1992) Proteins 12:42-48; and Lovejoy et al. (1994) Science 263:375-377. Thus, according to the similar metal dependency, at least one TRRE may be a part of the MMPs family of which 11 MMPs have been cloned.
The substrate-specificity of TRRE has been investigated using membrane receptors and antigens other than the two TNF-Rs. These receptors and antigens are expressed at sufficient levels on THP-1 cells to be detected by FACS analysis including (i) IL-1R, whose soluble form is known to be produced by proteolytic cleavage, (ii) CD30 (ki-1), which belongs to the same receptor family as TNF-R (TNF-R/NGF-R superfamily) and whose soluble form is produced presumably by a Zn2+-dependent metalloprotease, (iii) CD54 (ICAM1), which belongs to immunoglobulin superfamily of adhesion molecules including VCAM-1 and is known to have a soluble form, and (iv) CD11b, which belongs to the integrin family of adhesion molecules and which has not been shown to have a soluble form. TRRE is apparently very specific to only the cleavage of both TNF-Rs and did not affect any other membrane receptors and antigens which have soluble forms.
Given the involvement of TNF in a variety of pathological conditions, it would be desirable to identify and characterize factors that modulate expression of sequences encoding TRREs and/or which modulate activity of TRREs. The present invention relates to identification and characterization of such factors, as well as to methods of modulating TRRE activity.
The invention encompasses a composition which modulates TRRE activity. In one embodiment, the composition increases TRRE activity. In another embodiment, the composition decreases TRRE activity. In one embodiment, the composition further comprises a physiologically acceptable buffer.
In one embodiment of the present invention, the composition is encoded by a nucleic acid of at least 15 contiguous nucleotides of clones 2-8, 2-9, 2-14, 2-15, P2-2, P2-10, P2-13, P2-14, and P2-15, which are represented by SEQ ID NOs:1 to 10, or a complementary strand thereof. In another embodiment, the composition is an RNA encoded by at least 15 contiguous nucleotides of a sequence presented in any of SEQ ID NOs. 1 to 10, or a complementary strand thereof. The invention also encompasses nucleic acids encoding the amino acid sequences of at least 5 contiguous amino acids of any of SEQ ID NOs:147 to 154. In another embodiment, the composition is a protein encoded by at least 10 contiguous codons of a nucleic acid sequence presented in any of SEQ ID NOs. 1 to 10, or a complementary strand thereof
In another embodiment, the composition is an antisense nucleic acid that binds to a nucleic acid comprising at least 15 contiguous nucleotides of a nucleic acid sequence presented in any of SEQ ID NOs. 1 to 10, or a complementary strand thereof. In another embodiment, the composition is an antibody that binds to a protein encoded by at least 10 contiguous codons of any of SEQ ID NOs. 1 to 10, or a complementary strand thereof. In one embodiment, the composition further comprises a physiologically acceptable buffer.
In another embodiment, the invention encompasses a method of obtaining a composition which alters TRRE activity, comprising the steps of: introducing into a first cell with known TRRE activity clones from a library of a second cell with a different TRRE activity; selecting a first cell with altered TRRE activity; and isolating the clone from the first cell, wherein the clone encodes the composition. In one embodiment the method identifies clones which enhance TRRE activity, and in this case the TRRE activity of the first cell is higher than that of the second cell. In a variant of this method, the first and second cells are of the same cell type, and the change in TRRE activity can be caused by a change in the gene copy number; e.g., TRRE activity can increase if more copies of a gene encoding a factor that enhances expression of the TRRE are present, or TRRE activity can decrease if more copies of a gene encoding a factor which inhibits TRRE expression are present. In one embodiment the method identifies clones which decrease TRRE activity, and in this case the TRRE activity of the first cell is lower than that of the second cell. The invention further comprises a clone identified by this method.
In another embodiment, the invention encompasses a method of treating an individual having a disease associated with altered levels or activity of TNF comprising administering an amount of the composition which alters TRRE activity sufficient to indirectly or directly normalize said levels of TNF. In one embodiment, the disease is cancer. In various embodiments, the cancer is selected from the group consisting of astrocytoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neural ectodermal tumor, pancreatic ductal adenocarcinoma, small and large cell lung adenocarcinomas, squamous cell carcinoma, bronchoalveolarcarcinoma, epithelial adenocarcinoma and liver metastases thereof, hepatoma, cholangiocarcinoma, ductal and lobular adenocarcinoma, squamous and adenocarcinomas of the uterine cervix, uterine and ovarian epithelial carcinomas, prostatic adenocarcinomas, transitional squamous cell bladder carcinoma, B and T cell lymphomas (nodular and diffuse), plasmacytoma, acute and chronic leukemias, malignant melanoma, soft tissue sarcomas, and leiomyosarcomas. In one embodiment the disease is cachexia. In another embodiment the disease is an inflammatory disorder. In one embodiment the disease is selected from the group consisting of autoimmune diseases, endotoxin shock, rheumatoid arthritis, trauma, infection and multiple sclerosis. In one embodiment the method of administration is selected from the group consisting of locally, parenterally, subcutaneously, intramuscularly, intraperitoneally, intracavity, intrathecally, and intravenously.
In another embodiment, the invention encompasses a method of measuring the TNF-receptor releasing (TRRE) activity of a test protein, comprising the steps of: obtaining cells that do not express significant amounts of TNF-R (TNF-Rxe2x88x92 cells); manipulating the cells to express recombinant TNF-R (TNF-R+ cells); incubating the TNF-R+ cells in a suitable medium in the absence and presence of the protein; and measuring the level of soluble TNF-R in the cell supernatant, where the ratio of soluble TNF-R in the absence and presence of the protein is indicative of the TRRE activity of the protein. In another embodiment, the invention encompasses a protein with TRRE activity identified by this method.
In another embodiment, the invention encompasses a method of diagnosing a disease associated with altered levels or activity of the protein affecting TRRE activity, comprising the steps of: obtaining a biological sample from a patient; measuring activity of the protein in the sample; and comparing the activity to the activity of a control biological sample. In one embodiment the disease is cancer. In one embodiment the cancer is selected from the group consisting of glioblastoma, melanoma, neuroblastoma, adenocarcinoma, soft tissue sarcoma, leukemias, lymphomas and carcinoma. In one embodiment the cancer is carcinoma and is selected from the group consisting of astrocytoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neural ectodermal tumor, pancreatic ductal adenocarcinoma, small and large cell lung adenocarcinomas, squamous cell carcinoma, bronchoalveolarcarcinoma, epithelial adenocarcinoma and liver metastases thereof, hepatoma, cholangiocarcinoma, ductal and lobular adenocarcinoma, squamous and adenocarcinomas of the uterine cervix, uterine and ovarian epithelial carcinomas, prostatic adenocarcinomas, transitional squamous cell bladder carcinoma, B and T cell lymphomas (nodular and diffuse), plasmacytoma, acute and chronic leukemias, malignant melanoma, soft tissue sarcomas, and leiomyosarcomas.
In another embodiment, the invention encompasses a method of treating a disease associated with elevated levels of soluble TNF receptor comprising administering an amount of an inhibitor of TNF receptor releasing enzyme effective to decrease the levels of soluble TNF receptor. In another embodiment, the disease is cancer. In another embodiment, the cancer is selected from the group consisting of astrocytoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neural ectodermal tumor, pancreatic ductal adenocarcinoma, small and large cell lung adenocarcinomas, squamous cell carcinoma, bronchoalveolarcarcinoma, epithelial adenocarcinoma and liver metastases thereof, hepatoma, cholangiocarcinoma, ductal and lobular adenocarcinoma, squamous and adenocarcinomas of the uterine cervix, uterine and ovarian epithelial carcinomas, prostatic adenocarcinomas, transitional squamous cell bladder carcinoma, B and T cell lymphomas (nodular and diffuse), plasmacytoma, acute and chronic leukemias, malignant melanoma, soft tissue sarcomas, and leiomyosarcomas. In another embodiment, the inhibitor is selected from the group consisting of a metalloprotease inhibitor, an antibody that blocks the effective interaction between TNF receptor and TNF receptor releasing enzyme, a polynucleotide encoding said antibody, an antisense oligonucleotide specific for the gene encoding tumor necrosis receptor releasing enzyme, and a ribozyme specific for the gene encoding TNF receptor releasing enzyme. In another embodiment, the method further comprises the step of administering an amount of at least one cytokine effective to enhance an immune response against the cancer. In another embodiment, the cytokine is selected from the group consisting of interleukin 2, interleukin 4, granulocyte macrophage colony stimulating factor, and granulocyte colony stimulating factor. In another embodiment, the method further comprises the step of administering a chemotherapeutic agent. In another embodiment, the chemotherapeutic agent is selected from the group consisting of radioisotopes, vinca alkaloids, adriamycin, bleomycin sulfate, Carboplatin, cisplatin, cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin, Duanorubicin hydrochloride, Doxorubicin hydrochloride, Etoposide, fluorouracil, lomustine, mechlororethamine hydrochloride, melphalan, mercaptopurine, methotrexate, mitomycin, mitotane, pentostatin, pipobroman, procarbaze hydrochloride, streptozotocin, taxol, thioguanine, and uracil mustard.